The Role And Mechanism Of Cold Induced RNA Binding Protein In Injury Of Spermatogenic Cell Induced By Testicular Heat

Part 1 Differential target m RNA analysis and validation of downstream of CIRBP after testicular heat shock in mice[Purpose] Apoptosis and cell cycle arrest of spermatogenic cells in mice exposed to high temperature are related to the decrease of Cold-inducible RNA-binding protein(CIRBP)expression in spermatogenic cells of testis.However,the specific mechanism of CIRBP expression regulating spermatogenic cell apoptosis or cell cycle arrest has not been elucidated.Therefore,in the first part,the target m RNA downstream of CIRBP was sequenced before and after testicular heat shock,and bioinformatics analysis was carried out to find the key target m RNA and important pathway of testicular heat stress induced spermatogenesis damage.[Methods] 8-week-old male BALB / c mice were selected for testicular heat stress test at 43 ℃ for 30 minutes.Then,the testis was selected and the spermatogenic cells were purified for RNA immunoprecipitation and high-throughput sequencing(RIP-Seq).The sequencing results were screened by R language "limma" package,and the differential expressed genes(DEGs)were analyzed by bioinformatics.The important transcripts of CIRBP binding and the important pathway of spermatogenesis damage were found.The changes of key target m RNA were verified by RT-q PCR and agarose gel electrophoresis.[Results] Through bioinformatics analysis,780 DEGs were found in the downstream of CIRBP,including 503 up-DEGs and 277 down-DEGs.Through the analysis of GO enrichment,we found that they were enriched in cell cycle,RNA binding,splicing,transport.We found that they were mainly involved in several pathways through the analysis of KEGG,including Huntington’s disease,focal adhesion,spliceosome,protein processing in ER,RNA transport,etc.We found that the largest core protein cluster was related to cell cycle through PPI network analysis,including 23 proteins translated by core DEGs,and the most critical protein is cyclin B1(Ccnb1).Finaly,we found that the ability of CIRBP binding to Ccnb1 m RNA in testis increased about 1.9 times after heat stress.[Conclusions] The results domenstrated,It is obvious that the important transcripts of CIRBP binding was mainly involved in the process of cell cycle.and the difference of the binding ability of CIRBP and Ccnb1 m RNA was consistent with the results of high-throughput sequencing.Part 2 CIRBP regulates the expression of CCNB1 and the effect of heat shock on spermatogenic cell cycle[Purpose] To explore the effect of CIRBP on the regulation of CCNB1 and the resistance of CIRBP to the stagnation of spermatogenic cell cycle after heat shock in mice testis,to verify the target relationship of CIRBP on Ccnb1 m RNA,and to explore the function of CIRBP to resist the stagnation of spermatogenic cell cycle by regulating the expression of CCNB1 in mouse spermatocyte line(GC2-spd).[Methods] Firstly,the expression of CIRBP and CCNB1 in testis of mice before and after heat stress at 43 ℃ for 30 min was detected by Western blot,and the cycle arrest of spermatogenic cells was detected by flow cytometry.The direct binding of Ccnb1 m RNA to CIRBP was verified by RNA pulldown.Finally,si RNA interference silencing gene and lentivirus packaging plasmid overexpression gene were used to affect the expression of CIRBP in GC2-spd cells,and the regulatory effect of CIRBP on the expression of CCNB1 and the effect of CIRBP on the cycle arrest of spermatogenic cells.The expression changes of CIRBP and CCNB1 were detected by Western blot,and flow cytometry was used to detect whether CIRBP affects spermatogenic cell cycle arrest.[Results] It was found that the expression of CIRBP and CCNB1 protein changed after heat stress.The level of CIRBP protein decreased(-23.70%)at 3 h after induction,and decreased more at 6 h(-31.92%)and 9 h(-72.77%)after induction.There was no significant difference in the expression of CCNB1 after 3hours,which decreased(-32.32%)after 6 hours,lower than that of the control group,and continued to decrease(-38.47%)after 9 hours.Flow cytometry showed that the proportion of G1 phase and G2 / M phase increased under heat stress,suggesting that heat stress may lead to spermatogenic cell cycle arrest.RNA pulldown assay showed that the 3’UTR region of Ccnb1 m RNA was directly bound to CIRBP.Subsequently,the GC2-spd cell model of CIRBP proved that the expression of CCNB1 decreased with the silence of CIRBP expression,and increased with the overexpression of CIRBP.Flow cytometry showed that the decreased expression of CIRBP could enhance the proportion of G1 phase and G2 / M phase of GC-2spd cells;the over expression of CIRBP could reduce the proportion of G1 phase and G2 / M phase,and increase the proportion of S phase.It is suggested that CIRBP is related to the cell cycle of GC2-spd.[Conclusions] After testicular heat shock,the expression level of CCNB1 decreased with the decrease of CIRBP expression level.CIRBP binds to the 3’UTR region of Ccnb1 m RNA and regulates the expression of CCNB1,and affects the cell cycle of spermatogenic cells.